Six et al. Parasites & Vectors (2016) 9:90 DOI 10.1186/s13071-016-1372-1

RESEARCH Open Access Comparative speed of kill of sarolaner (Simparica™) and afoxolaner (NexGard®) against induced infestations of Ctenocephalides felis on dogs Robert H. Six1*, Julian Liebenberg2, Nicole A. Honsberger1 and Sean P. Mahabir1

Abstract Background: Fleas are the most common ectoparasite infesting dogs globally. The many possible sequellae of infestation include: direct discomfort; allergic reactions; and the transmission of pathogens. Rapid speed of kill is an important characteristic for a parasiticide in order to alleviate the direct deleterious effects of fleas, reduce the impact of allergic responses, and break the flea infestation cycle. In this study, the speed of kill of a novel orally administered isoxazoline parasiticide, sarolaner (Simparica™) against fleas on dogs was evaluated and compared with afoxolaner (NexGard®) for 5 weeks after a single oral dose. Methods: Twenty-four dogs were randomly allocated to treatment with a single oral dose at label rate of either sarolaner (2 to 4 mg/kg) or afoxolaner (2.5 to 6.8 mg/kg) or placebo, based on pretreatment flea counts. Dogs were combed and live fleas counted at 8, 12 and 24 h after treatment and subsequent re-infestations on Days 7, 14, 21, 28 and 35. Efficacy was determined at each time point relative to counts for placebo dogs. Results: There were no adverse reactions to treatment. A single oral dose of sarolaner provided ≥98.8 % efficacy (based on geometric means) within 8 h of treatment or subsequent weekly re-infestations of fleas to Day 35. By 12 h, fleas were virtually eradicated from all dogs, with only two fleas recovered from a single sarolaner-treated dog on Day 7; efficacy was 100 % at all other time points. Significantly greater numbers of live fleas were recovered from afoxolaner-treated dogs at 8 h on all days and at 12 h on Days 28 and 35 (P < 0.05). Conclusions: In this controlled laboratory evaluation, sarolaner had a significantly faster speed of kill against fleas than afoxolaner. This was noticeably more evident towards the end of the treatment period. The rapid and consistent kill of fleas within 8 to 12 h after a single oral dose of sarolaner over 35 days indicates that this treatment will provide highly effective control of flea infestations, relief for dogs afflicted with flea allergy dermatitis, and should reduce the risk of flea-borne pathogen transmission. Keywords: Ctenocephalides felis, Sarolaner, Simparica™, Afoxolaner, Speed of kill, Flea, Dog, Oral

* Correspondence: [email protected] 1Zoetis, Veterinary Medicine Research and Development, 333 Portage St., Kalamazoo, MI 49007, USA Full list of author information is available at the end of the article

© 2016 Six et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Six et al. Parasites & Vectors (2016) 9:90 Page 2 of 6

Background Methods Flea infestation of pets and the home are a common oc- Ethical approval currence and their control and elimination can become The study was a masked, negative controlled, randomized, an expensive and time consuming challenge [1]. The cat laboratory efficacy design conducted in the Republic of flea, Ctenocephalides felis felis, is the most abundant South Africa. Procedures were in accordance with the ectoparasite of dogs and cats worldwide [2], and they World Association for the Advancement of Veterinary cause flea allergic dermatitis (FAD), serve as the vector of Parasitology (WAAVP) guidelines for evaluating the effi- various bacterial pathogens, and are the intermediate host cacy of parasiticides for the treatment, prevention and for filarid and cestode parasites. FAD has been recognized control of flea and tick infestation on dogs and cats [16] as the most common dermatologic disease of dogs [3]. and complied with the principals of Good Clinical Ctenocephalides felis has been implicated in the transmis- Practices [17]. The protocol was reviewed and approved by sion of Rickettsia typhi, Rickettsia felis, Bartonella hense- the local Institutional Animal Care and Use Committee. lae, Mycoplasma haemofelis and, in rare cases, Yersinia Masking of the study was assured through the separation of pestis [4–7]. Ctenocephalides felis also serves as the inter- functions. All personnel conducting observations or animal mediate host for the nonpathogenic subcutaneous filarid care or performing infestations and counts were masked to nematode of dogs, Acanthocheilonema (Dipetalonema) treatment allocation. reconditum, as well as several species of cestodes includ- ing Dipylidium caninum and Hymenoleptis spp. [1, 8, 9]. Animals Effective control of fleas and their associated condi- Twenty-four, male and female, purpose-bred mongrel tions and diseases is dependent upon the rapid removal dogs ranging in age from 15 to 105 months and weigh- of live fleas from the host. Fleas feed almost immediately ing from 7.8 to 21.0 kg were used in the study. All dogs on attaining a host [10], and direct irritation and allergic had undergone an adequate wash-out period to ensure reactions are dependent upon the frequency and dur- that no residual ectoparasiticide efficacy remained from ation of feeding [11]. Similarly, the longer that a host is any previously administered compound. Each dog was exposed to feeding fleas the greater the chance of the individually identified by electronic transponder. The transmission of flea-borne pathogens. The use of highly dogs were acclimatized to study conditions for a mini- effective, topical and systemic parasiticides has allowed mum of 14 days before treatment on Day 0. Dogs were in- theprimarymeansoffleacontroltobeviadirect dividually housed in indoor runs such that no physical treatment of the pet. These treatments have largely contact was possible between dogs. Dogs were fed an ap- eliminated the need to treat indoor and outdoor envi- propriate maintenance ration of a commercial dry canine ronments, and their use as host targeted therapies feed, and water was available ad libitum. All dogs were markedly reduces the severity and prevalence of FAD given a physical examination to evaluate general health [2]. Orally administered compounds, whether adminis- and suitability for inclusion into the study. General health tered daily like nitenpyram [12] or monthly like spino- observations were performed twice daily from the start of sad [13] have been introduced that provide rapid the acclimation period to the end of the study. systemic control of fleas for immediate daily treatment, nitenpyram [12], or for up to a month following a sin- Design gle dose, spinosad [13]. These applications have been The study followed a randomized complete block design. readily accepted by veterinarians and dog owners for Dogs were ranked according to decreasing flea counts their efficacy, ease of use and the fact that these sys- into blocks of three and within each block a dog was temic agents are not topically applied with the resultant randomly allocated to treatment with placebo, sarolaner, lowerexposureoftheownerorchildrentopotentially or afoxolaner. There were eight dogs per treatment toxic residues. Recently, a new class of compounds, the group. Dogs were infested with fleas prior to treatment isoxazolines, have shown excellent efficacy against both and then weekly following treatment for 5 weeks. Flea fleas and ticks for a month or longer following a single counts were conducted at 8, 12 and 24 h after treatment orally administered dose [14, 15]. and each subsequent weekly re-infestation. ™ Sarolaner (Simparica , Zoetis) is a new isoxazoline effective against fleas and ticks for at least one month Treatment following a single dose. A laboratory study was con- Day -1 bodyweights were used to determine the appro- ducted to compare the speed of kill of a single dose of priate dose to be administered. On Day 0, dogs received ™ sarolaner (Simparica , Zoetis) and afoxolaner (NexGard®, either a placebo tablet, a sarolaner chewable tablet ™ Merial) against an existing flea (C. felis) infestation and (Simparica ) to deliver sarolaner at the minimum label subsequent re-infestations for a period of 5 weeks after dose of 2 mg/kg (range 2 to 4 mg/kg), or NexGard® per treatment. label directions (afoxolaner at 2.5 to 6.8 mg/kg). All Six et al. Parasites & Vectors (2016) 9:90 Page 3 of 6

doses were administered by hand pilling to ensure ac- flea counts relative to placebo calculated using Abbott’s curate and complete dosing. Each dog was observed for formula: several minutes after dosing for evidence that the dose mean countðÞ placebo –mean countðÞ treated was swallowed, and for general health at 1, 4 and 24 h % reduction ¼ 100 Â mean countðÞ placebo after treatment administration.

Flea infestation and assessment Results The C. felis used in the study were from a locally main- There were no treatment-related adverse events ob- tained laboratory colony of European origin. The colony served during the study. Placebo-treated dogs main- was initiated in 2010 with fleas from Germany; fleas ob- tained good flea infestations throughout the study and tained from Ireland were introduced into the colony in these counts were maintained even following the comb- 2012. Flea infestations were performed on Days -7 (host ing and re-infestation procedures at 8 and 12 h (Tables 1, suitability and allocation), -1, 7, 14, 21, 28 and 35. At 2 and 3). each infestation a precounted aliquot of 100 (±5) adult, At the 8-h time point, both treatments resulted in sig- unfed C. felis were directly applied to the animal which nificantly lower flea counts than placebo-treated dogs was gently restrained for a few minutes to allow the fleas (P ≤ 0.0005) throughout the study (Table 1). Treatment to penetrate and disperse into the hair coat. Each dog with sarolaner also resulted in significantly lower flea was examined and combed to remove and count fleas at counts than afoxolaner at 8 h on all assessment days 24 h after the initial host suitability infestation, and at 8 (P ≤ 0.0499) and the sarolaner treatment provided greater (±1), 12 (±1) and 24 (±2) hours after treatment and each and more consistent efficacy at 8 h (≥98.8 %, ≥97.9 % geo- subsequent weekly re-infestation. Fleas were replaced on metric, arithmetic mean) for the entire study. Efficacy the dogs immediately after each 8 and 12 h evaluation, based on geometric means for afoxolaner ranged from and discarded after the 24 h counts. 76.3 to 99.4 %, and on arithmetic means from 65.2 to Flea counts were performed by personnel trained in the 99.1 % (Table 1). Notably, the efficacy for the afoxolaner- standard procedures in use at the test facility. Commercial treated dogs declined as the study progressed, and fine-toothed flea combs were used. Dogs were combed was <90 % from Day 21 onwards (Fig. 1). using repeated strokes initially while standing starting At the 12-h timepoint, both treatments resulted in sig- from the head, then proceeding caudally along the dor- nificantly lower flea counts than placebo-treated dogs sum. The dog was then placed on each side and then on (P ≤ 0.0001) throughout the study (Table 2) and flea its back for combing of the sides and ventral surfaces. counts were significantly higher for afoxolaner-treated After a few combing strokes were completed, the comb dogs on Days 28 and 35 (P ≤ 0.0161). Efficacy was very was examined and hair and fleas were removed from the high for sarolaner with only two live fleas found on one comb and all live fleas were counted. Each animal was dog on Day 7 (efficacy of 99.6 %); on all other days efficacy examined for a minimum of 10 min; if any fleas were re- for sarolaner was 100 %. For afoxolaner, 100 % efficacy covered in the last minute, combing was continued in was only achieved on Day 0, and efficacy tended to decline one-minute increments until no fleas were detected. as the study progressed (Fig. 1). On Days 28 and 35, effi- cacy for afoxolaner-treated dogs was <95 % (arithmetic Statistical analysis mean) (Table 2). The individual dog was the experimental unit and the At the 24-h time point, both treatments resulted in primary endpoint was the live flea count. Data for post- significantly lower flea counts than placebo-treated dogs treatment flea counts were summarized with arithmetic (P ≤ 0.0001) throughout the study (Table 3). Both prod- and geometric means by treatment group and timepoint. ucts provided ≥99.8 % reduction in mean live flea counts Flea counts were transformed (loge(count + 1)) prior to through Day 35 with 100 % efficacy at most evaluations analysis in order to stabilize the variance and normalize (Table 3); there was no difference between the counts the data. Using the PROC MIXED procedure (SAS 9.2, for the two products on any assessment day (P ≥ 0.5043). Cary NC), transformed counts were analyzed using a mixed linear model. The fixed effects were treatment, Discussion timepoint and the interaction between timepoint and For any parasiticide product providing flea control, the treatment by timepoint. The random effects included product’s initial speed of kill is important to provide the room, block within room, block by treatment interaction pet with rapid relief from the existing infestation. The within room, and error. Testing was two-sided at the residual speed of kill is critical to decrease the likelihood significance level α = 0.05. of transmission of vector borne disease, ensure that any The assessment of efficacy was based on the percent newly acquired fleas are rapidly eliminated before they reduction in the arithmetic and geometric mean live can reproduce, and assist in the management of flea Six et al. Parasites & Vectors (2016) 9:90 Page 4 of 6

Table 1 Mean live flea counts and efficacy relative to placebo at 8 h after treatment and post treatment re-infestations for dogs treated with a single oral dose of sarolaner or afoxolaner on Day 0 Treatment Day of treatment or re-infestation 0 7 14212835 Placebo Range 44–99 51–91 69–89 55–92 62–97 75–100 A. mean 69.9 74.9 77.0 79.8 79.1 87.5 G. mean1 68.1a 73.3a 76.6a 78.8a 78.4a 87.1a Sarolaner Range 0–00–30–00–00–80–5 A. mean 0.0 0.4 0.0 0.0 1.6 0.6 Efficacy (%) 100 99.5 100 100 97.9 99.3 G. mean1 0.0c 0.2c 0.0c 0.0c 1.0c 0.3c Efficacy (%) 100 99.7 100 100 98.8 99.7 P-value vs. placebo <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 Afoxolaner Range 0–20–35 0–31 0–44 3–66 0–71 A. mean 0.6 6.1 10.1 15.3 27.5 25.0 Efficacy (%) 99.1 91.8 86.9 80.9 65.2 71.4 G. mean1 0.4b 2.4 b 6.8b 8.5b 18.6b 11.3b Efficacy (%) 99.4 96.7 91.1 89.2 76.3 87.0 P-value vs. placebo <0.0001 <0.0001 <0.0001 <0.0001 0.0005 0.0005 P-value vs. sarolaner 0.0499 0.0094 <0.0001 <0.0001 <0.0001 <0.0001 1Geometric means within columns with the same superscript are not significantly different (P > 0.05)

Table 2 Mean live flea counts and efficacy relative to placebo at 12 h after treatment and post treatment re-infestations for dogs treated with a single oral dose of sarolaner or afoxolaner on Day 0 Treatment Day of treatment or re-infestation 0 7 14212835 Placebo Range 40–95 33–79 60–89 48–88 56–85 62–95 A. mean 62.3 59.5 74.6 75.8 70.0 80.4 G. mean1 60.4a 57.0a 74.1a 74.7a 69.3a 79.8a Sarolaner Range 0–00–20–00–00–00–0 A. mean 0.0 0.3 0.0 0.0 0.0 0.0 Efficacy (%) 100 99.6 100 100 100 100 G. mean1 0.0b 0.1b 0.0b 0.0b 0.0c 0.0c Efficacy (%) 100 99.7 100 100 100 100 P-value vs.placebo <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 Afoxolaner Range 0–00–20–20–40–28 0–25 A. mean 0.0 0.3 0.3 0.9 5.5 4.3 Efficacy (%) 100 99.6 99.7 98.8 92.1 94.7 G. mean1 0.0b 0.1b 0.1b 0.5b 2.5b 1.4b Efficacy (%) 100 99.7 99.8 99.4 96.4 98.2 P-value vs. placebo <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 P-value vs. sarolaner 1.000 1.000 0.2455 0.0713 0.0008 0.0161 1Geometric means within columns with the same superscript are not significantly different (P > 0.05) Six et al. Parasites & Vectors (2016) 9:90 Page 5 of 6

Table 3 Mean live flea counts and efficacy relative to placebo at 24 h after treatment and post treatment re-infestations for dogs treated with a single oral dose of sarolaner or afoxolaner on Day 0 Treatment Day of treatment or re-infestation 0 7 14212835 Placebo Range 36–87 28–88 48–87 53–73 43–73 58–83 A. mean 56.5 59.3 64.3 66.3 59.8 71.4 G. mean1 54.6a 56.6a 62.7a 66.0a 59.0a 70.8a Sarolaner Range 0–00–10–00–00–00–0 A. mean 0.0 0.1 0.0 0.0 0.0 0.0 Efficacy (%) 100 99.8 100 100 100 100 G. mean1 0.0b 0.1b 0.0b 0.0b 0.0b 0.0b Efficacy (%) 100 99.8 100 100 100 100 P-value vs.placebo <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 Afoxolaner Range 0–00–00–00–00–00–0 A. mean 0.0 0.0 0.0 0.0 0.0 0.0 Efficacy (%) 100 100 100 100 100 100 G. mean1 0.0b 0.0b 0.0b 0.0b 0.0b 0.0b Efficacy (%) 100 100 100 100 100 100 P-value vs. placebo <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 P-value vs. sarolaner 1.000 0.5043 1.000 1.000 1.000 1.000 1Geometric means within columns with the same superscript are not significantly different (P > 0.05) allergic dermatitis. The added benefit for products pre- control of greater than 97 % within 8 h after treatment scribed by a veterinarian is instilling pet owner confidence and weekly re-infestations for 35 days, and fleas were vir- in the outcome of the treatment and the treatment recom- tually eliminated from all dogs within 12 h over the entire mendation which can enhance compliance. A single oral 35 days. This rapid efficacy will disrupt the flea life cycle dose of sarolaner at the proposed commercial dose of 2 to by killing adult fleas before they can lay eggs, thus redu- 4 mg/kg resulted in the rapid reduction of an existing flea cing the environmental infestation level [18, 19]. infestation as well as rapid kill of newly infested fleas for The efficacy provided by sarolaner at 8 h was signifi- 35 days. Treatment with sarolaner resulted in effective cantly greater than that provided by afoxolaner at all

Fig. 1 Percent efficacy based on geometric mean counts relative to placebo at 8 and 12 h after treatment and weekly post treatment re-infestations of fleas for dogs treated with a single oral dose of sarolaner or afoxolaner on Day 0 Six et al. Parasites & Vectors (2016) 9:90 Page 6 of 6

evaluations but especially when performance is com- Received: 4 December 2015 Accepted: 16 January 2016 pared towards the end of the monthly treatment period when the efficacy of afoxolaner declined below 90 % References from Day 21 onwards. Efficacy of afoxolaner was im- 1. Blagburn BL, Dryden MW. Biology, treatment and control of flea and tick proved at 12 h and again sarolaner had consistently bet- infestations. Vet Clin Small Anim. 2009;39:1173–200. 2. Rust MK. Advances in the control of Ctenocephalides felis felis (cat flea) on ter kill of fleas and there were significantly more live cats and dogs. Trends Parasitol. 2005;21:232–6. fleas recovered from the afoxolaner-treated dogs on 3. Scott DW, Miller WH, Griffin CE. Muller and Kirk’s small animal dermatology. Days 28 and 35; sarolaner was 100 % effective on these Philadelphia: Saunders; 2001. p. 626. 4. Breitschwerdt EB. Feline bartonellosis and cat scratch disease. Vet Immunol days. 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Author details 1Zoetis, Veterinary Medicine Research and Development, 333 Portage St., Kalamazoo, MI 49007, USA. 2ClinVet International (pty) Ltd, Uitsigweg, Bainsvlei 9338, Bloemfontein, Republic of South Africa.